Tumor Therapy with an Antibody for Vascular Endothelial Growth Factor and an Antibody for Human Epithelial Growth Factor Receptor Type 2

ABSTRACT

The present invention provides a method of treating a breast cancer disease in a patient who has failed prior treatment with an anti-VEGF antibody, comprising administering to the patient a therapeutically effective amount of an anti-HER2 antibody while continuing said anti-VEGF antibody therapy. The invention also provides corresponding pharmaceutical kits and pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/947,264, filed Nov. 16, 2010, which is a continuation of U.S. patentapplication Ser. No. 11/725,777, filed Mar. 20, 2007, which claims thebenefit of European Patent Application No. EP06111523.4, filed Mar. 22,2006, and European Patent Application No. EP06021815.3, filed Oct. 18,2006, all of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention is directed to combined therapy with anti-HER2 andanti-VEGF antibodies. In particular, the invention concerns the use ofsuch antibodies to treat breast cancer disease in a patient who hasfailed prior breast cancer treatment with an anti-VEGF antibody.

BACKGROUND OF THE INVENTION

Angiogenesis is implicated in the pathogenesis of a variety of disorderswhich include solid tumors, intraocular neovascular syndromes such asproliferative retinopathies or age-related macular degeneration (AMD),rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev. Physiol. 53(1991) 217-239; and Garner, A, Vascular diseases, In: Pathobiology ofocular disease, A dynamic approach, (eds.) Garner and A, Klintworth, GK, 2nd Edition Marcel Dekker, New York, (1994), pp 1625-1710). In thecase of solid tumors, the neovascularization allows the tumor cells toacquire a growth advantage and proliferative autonomy compared to thenormal cells. Accordingly, a correlation has been observed betweendensity of microvessels in tumor sections and patient survival in breastcancer as well as in several other tumors (Weidner, N., et al., N. Engl.J. Med. 324 (1991) 1-6; Horak, E. R., et al., Lancet 340 (1992)1120-1124; and Macchiarini, P., et al., Lancet 340 (1992) 145-146).

Vascular endothelial growth factor (VEGF) is involved in the regulationof normal and abnormal angiogenesis and neovascularization associatedwith tumors and intraocular disorders (Ferrara, N., et al., Endocr. Rev.18 (1997) 4-25; Berkman, R. A., et al., J. Clin. Invest. 91 (1993)153-159; Brown, L. F., et al., Human Pathol. 26 (1995) 86-91; Brown, L.F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit.J. Cancer 73 (1996) 931-934; and Dvorak, H. F., et al., Am. J. Pathol.146 (1995) 1029-1039). Anti-VEGF neutralizing antibodies suppress thegrowth of a variety of human tumor cell lines in mice (Kim, K. J., etal., Nature 362 (1993) 841-844; Warren, R. S., et al., J. Clin. Invest.95 (1995) 1789-1797; Borgstrom, P., et al., Cancer Res. 56 (1996)4032-4039; and Melnyk, O., et al., Cancer Res. 56 (1996) 921-924).WO94/10202, WO 98/45332, WO 2005/00900 and WO00/35956 refer to antibodiesagainst VEGF. Humanized monoclonal antibody bevacizumab (sold under thetradename Avastin®) is an anti-VEGF antibody used in tumor therapy andis the only anti-angiogenic agent approved for treatment of cancer (WO98/45331).

HER2 is a member of the human epidermal growth factor receptor familyand possesses protein kinase activity in its cytoplasmic domain. HER2 isover-expressed in tumor cells and is correlated with poor prognosis andsurvival. HER2 is therefore a valuable target of breast cancer therapy.Antibodies against HER2 are known from Takai, N., et al., Cancer 104(2005) 2701-2708; Yeon, C. H., et al., Invest. New Drugs 23 (2005)391-409; Wong, W. M., et al., Cancer Pract. 7 (1999) 48-50; Albanell,J., et al., Drugs Today (Barc). 35 (1999) 931-46.

Trastuzumab (sold under the tradename Herceptin®) is a recombinanthumanized anti-HER2 monoclonal antibody used for the treatment of HER2over-expressed/HER2 gene amplified metastatic breast cancer. Preclinicalstudies demonstrated that the antibody has anti-tumor activity in vivoand in vitro. Moreover, additive or synergistic enhancement ofanti-tumor activity of trastuzumab was observed in combination withvarious anti-tumor agents in mouse models. In clinical studies,extension of survival was observed in HER2 overexpressing metastaticbreast cancer patients.

According to WO 98/45331, the effectiveness of an anti-VEGF antibody inpreventing or treating disease may be improved by administering theantibody serially or in combination with another agent that is effectivefor those purposes, such as an antibody capable of binding to HER2receptor. WO 2005/012531 describes antibodies that may be combined withan anti-VEGF antibody (e.g. Avastin®) and/or anti-ErbB antibodies (e.g.Herceptin®) in the treatment of colorectal cancer, metastatic breastcancer and kidney cancer. According to WO 2005/063816, anti-VEGFantibodies may be combined with anti-ErbB antibodies in a treatment ofmetastatic breast cancer. WO 2005/00090 and WO 2003/077841 also disclosethe combination of anti-VEGF antibodies with anti-ErbB2 antibodies fortumor therapy.

Clinical oncologists are in agreement that the failure of cancertreatment is not necessarily caused by the growth of the primary tumor,which is generally dealt with using surgery, but rather by themetastatic spread into different organs. The regression of primarytumors by different cytotoxic drugs is not always indicative foranti-metastatic activity per se. On the contrary, enhanced metastasishas been observed in response to several anti-cancer drugs (Geldof, A.A., et al., Anticancer Res. 8 (1988) 1335-1339; Murphy, S. B., J. Clin.Oncol. 11 (1993) 199-201; and De Larco, J. E., et al., Cancer Res. 61(2001) 2857-2861). Clearly there exists a need to develop treatmenttherapies that target not only the primary tumor, but also suppressmetastasis.

SUMMARY OF THE INVENTION

The invention comprises the use of an anti-HER2 antibody and ananti-VEGF antibody for the manufacture of a medicament for treating abreast cancer disease in a patient who has failed prior cancer therapywith an anti-VEGF antibody, comprising administering to the patient atherapeutically effective amount of an anti-HER2 antibody and ananti-VEGF antibody.

In a preferred embodiment, the invention comprises the use oftrastuzumab and bevacizumab for the manufacture of a medicament fortreating a breast cancer disease characterized by an overexpression ofthe HER2 receptor protein in a patient who has failed prior therapy withan anti-VEGF antibody such as bevacizumab, comprising administering tothe patient a therapeutically effective amount of trastuzumab andbevacizumab.

The invention further comprises a method of treating a breast cancerdisease in a patient who has failed prior therapy with an anti-VEGFantibody, comprising administering to the patient a therapeuticallyeffective amount of an anti-HER2 antibody while continuing saidanti-VEGF antibody therapy.

The invention further comprises a method of treating a breast cancerdisease, in a patient who has failed prior therapy with an anti-VEGFantibody, comprising administering to the patient a therapeuticallyeffective amount of trastuzumab while continuing bevacizumab therapy,wherein the breast cancer disease is characterized by an overexpressionof the HER2 receptor protein.

The invention further comprises a method for increasing the duration ofsurvival of a patient having breast cancer disease who has failed priortherapy with an anti-VEGF antibody, comprising administering to thepatient effective amounts of an anti-VEGF antibody and an anti-HER2antibody, whereby the co-administration of the anti-VEGF antibody andthe anti-HER2 antibody effectively increases the duration of survival.

The invention further comprises a method for increasing the progressionfree survival of a patient having breast cancer disease who has failedprior therapy with an anti-VEGF antibody, comprising administering tothe patient effective amounts of an anti-VEGF antibody and an anti-HER2antibody, whereby the co-administration of the anti-VEGF antibody andthe anti-HER2 antibody effectively increases the duration of progressionfree survival.

The invention further comprises a method for treating a group ofpatients, having breast cancer disease and having failed prior therapywith an anti-VEGF antibody, comprising administering to the patienteffective amounts of an anti-VEGF antibody and an anti-HER2 antibody,whereby the co-administration of the anti-VEGF antibody and theanti-HER2 antibody effectively increases the response rate in the groupof patients.

The invention further comprises a method for increasing the duration ofresponse of a patient having breast cancer disease who has failed priortherapy with an anti-VEGF antibody, comprising administering to thepatient effective amounts of an anti-VEGF antibody and an anti-HER2antibody, whereby the co-administration of the anti-VEGF antibody andthe anti-HER2 antibody effectively increases the duration of response.

The invention further comprises a method of treating a patient havingbreast cancer disease who has failed prior therapy with an anti-VEGFantibody, comprising administering to the patient effective amounts ofan anti-VEGF antibody and an anti-HER2 antibody, whereby theco-administration of the anti-VEGF antibody and the anti-HER2 antibodyresults in statistically significant and clinically meaningfulimprovement of the treated patient as measured by the duration ofsurvival, progression free survival, response rate or duration ofresponse.

This invention further comprises a method for reducing metastasis in apatient having breast cancer disease who has failed prior therapy withan anti-VEGF antibody, comprising administering to the patient effectiveamounts of an anti-VEGF antibody and an anti-HER2 antibody, whereby theco-administration of the anti-VEGF antibody and the anti-HER2 antibodyeffectively reduces metastasis.

The invention further comprises a method for treating a group ofpatients, having breast cancer disease and having failed prior therapy,with an anti-VEGF antibody, comprising administering to the patienteffective amounts of an anti-VEGF antibody and an anti-HER2 antibody,whereby the co-administration of the anti-VEGF antibody and theanti-HER2 antibody effectively reduces metastasis in the group ofpatients.

The invention provides an article of manufacture (e.g., pharmaceuticalkit) comprising one or more containers, and preferably at least twocontainers, a pharmaceutical composition within a first containercomprising an anti-VEGF antibody, a pharmaceutical composition within asecond container comprising an anti-HER2 antibody and a package insertinstructing the user of the composition to administer to a patient,having breast cancer disease who has failed prior therapy with ananti-VEGF antibody, the anti-VEGF antibody within said first containerand an anti-HER2 antibody within said second container.

The invention further provides for a pharmaceutical compositioncomprising an anti-HER2 antibody and an anti-VEGF antibody useful in thetreatment of breast cancer disease in a patient which has failed priortherapy with an anti-VEGF antibody. Preferably the anti-HER2 antibody istrastuzumab. Also preferably the anti-VEGF antibody is bevacizumab.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Antitumor activity of combined trastuzumab and bevacizumabtreatment on tumor growth after bevacizumab treatment failure. Meanvalues of tumor volume (mm³) plotted on the y-axis; number of days afterinjection of tumor cells plotted on the x-axis. Vehicle (circles),trastuzumab at loading dose of 30 mg/kg and maintenance dose of 15 mg/kg(squares), bevacizumab at 5 mg/kg until day 55 when treatment alsoincludes trastuzumab at 15 mg/kg (triangles).

FIG. 2 Effect of combined trastuzumab and bevacizumab treatment on lungmetastasis. Mean value of human Alu DNA sequence (ng/ml) quantitatedfrom lung tissue using real-time PCR and plotted on the y-axis.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated by reference intheir entirety.

Definitions

The term “VEGF” according to the invention refers to the vascularendothelial cell growth factor (Swiss-Prot No. P 15692), alternativesplicing forms (see e.g. Leung, D. W., et al., Science, 246 (1989)1306-1309; and Houck, K. A., et al., Mol. Endocrin. 5 (1991) 1806-1814)and active fragments, preferably N-terminal fragments thereof.

The term “anti-VEGF antibody” according to the invention is an antibodythat binds specifically to VEGF. The preferred humanized anti-VEGFantibody or variant anti-VEGF antibody herein binds human VEGF with a Kdvalue of no more than about 1×10⁻⁸M and preferably no more than about5×10⁻⁹M. Preferably the anti-VEGF antibody is a monoclonal antibody thatbinds to the same epitope as recombinant humanized anti-VEGF monoclonalantibody generated according to Presta, L. G., et al., Cancer Res. 57(1997) 4593-4599. A preferred antibody is bevacizumab. Anti-VEGFantibodies and methods for their manufacture are e.g. described in U.S.Pat. No. 6,054,297, US 2003/0190317, U.S. Pat. No. 6,632,926, US6,884,879, and US 2005/0112126.

Bevacizumab comprises mutated human IgG1 framework regions andantigen-binding complementarity-determining regions from a murineanti-hVEGF monoclonal antibody that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 Daltons and isglycosylated. Bevacizumab and its method of preparation are described inEP 1 325 932.

HER2 is a 185-kDa growth factor receptor also referred to as neu andc-erbB-2 (Slamon, D. J., et al., Science 235 (1987) 177-182; Swiss-ProtP04626) whose function is related to neoplastic transformation in humanbreast cancer cells. Overexpression of this protein has been identifiedin 20-30% of breast cancer patients where it correlates with regionallyadvanced disease, increased probability of tumor recurrence, and reducedpatient survival. As many as 30-40% of patients having gastric,endometrial, salivary gland, non-small cell lung, pancreatic, ovarian,peritoneal, prostate, or colorectal cancers may also exhibitoverexpression of this protein. Anti-HER2 antibodies and methods fortheir manufacture are e.g. described in U.S. Pat. No. 6,054,297, WO89/06692, U.S. Pat. No. 6,953,842, U.S. Pat. No. 6,949,245, U.S. Pat.No. 6,399,063, U.S. Pat. No. 6,165,464, U.S. Pat. No. 6,054,297, U.S.Pat. No. 5,772,997, WO 2003/087131, WO 01/00245, WO 01/00238, WO00/69460, WO 00/52054, WO 99/31140 and WO 98/17797. In a preferredembodiment of the invention, the anti-HER2 antibody is trastuzumab.Trastuzumab and its method of preparation are described in EP 0 590 058.

The term “overexpression” of the HER2 receptor protein is intended toindicate an abnormal level of expression of the HER2 receptor protein ina cell from a tumor within a specific tissue or organ of the patientrelative to the level of expression in a normal cell from that tissue ororgan. Patients having a cancer characterized by overexpression of theHER2 receptor can be determined by standard assays known in the art.Preferably overexpression is measured in fixed cells of frozen orparaffin-embedded tissue sections using immunohistochemical (IHC)detection. When coupled with histological staining, localization of thetargeted protein can be determined and extent of its expression within atumor can be measured both qualitatively and semi-quantitatively. SuchIHC detection assays are known in the art and include the Clinical TrialAssay (CTA), the commercially available LabCorp 4D5 test, and thecommercially available DAKO HercepTest®(DAKO, Carpinteria, Calif.). Thelatter assay uses a specific range of 0 to 3+ cell staining (0 beingnormal expression, 3+ indicating the strongest positive expression) toidentify cancers having overexpression of the HER2 protein (see theHerceptin®(trastuzumab) full prescribing information, September 1998,Genentech Inc., San Francisco, Calif.). Thus, patients having a cancercharacterized by overexpression of the HER2 protein in the range of 1+,2+, or 3+, preferably 2+ or 3+, more preferably 3+ would benefit fromthe methods of therapy of the present invention.

The term “breast cancer disease” refers to the uncontrolled growth ofabnormal breast cells. It includes ductal carcinoma in situ, invasiveductal carcinoma, lobular carcinoma in situ, invasive lobular carcinoma,medullary carcinoma, Paget's disease of the nipple and metastatic breastcancer, as well as other cancer diseases of the breast as known to oneof ordinary skill in the art.

The term “failed prior therapy with an anti-VEGF antibody” or “treatmentfailure” as used herein refers to tumor patients who failed to respondto previous therapy with an anti-VEGF antibody (“non- responders”) orwho initially responded to previous therapy, but in whom the therapeuticresponse was not maintained (referred to as “relapsers”). Preferably theterm “failed prior therapy with an anti-VEGF antibody” refers torelapsers. Treatment failure (respectively Response (RE) andNon-Response (NR)) is established based on the medical judgment of apractitioner ascertained by the results from clinical and laboratorydata that are generally known in the art to assess patient treatment.Such data may be obtained, by way of example, from clinical examination,cytological and histological techniques, endoscopy and laparoscopy,ultrasound, CT, PET and MRI scans, chest X-ray and mammography, andmeasuring the concentration of tumor markers, such as CEA, Cyfra,CA15-3, interleukin 8 and soluble HER2. In this context “treatmentfailure” is defined as the absence of clinical improvement.Alternatively, RECIST criteria may be used to determine tumor response(Therasse, P., et al., J. Nat. Cancer Institute 92 (2000) 205-216) Inthis context “treatment failure” is defined as either “incompleteresponse/stable disease” or “progressive disease”.

According to these RECIST criteria, tumor response for solid tumors(Therasse, P., et al., J. Nat. Cancer Institute 92 (2000) 205-216) iscategorized in dependency of the volume progression or regression of thetumors (e.g. measured via CT) into four levels: complete response (CR)or partial response (PR), stable disease (SD) and progressive disease(PD) (see Table 1). Furthermore the European Organization for Researchand Treatment of Cancer (EORTC) proposed a categorization into fourlevels in dependency of the metabolism of the tumors measured via2-[¹⁸F]-Fluoro-2-deoxyglucose positron emission tomography (FDG-PET)(Young H., et al., Eur. J. Cancer 35 (1999) 1773-1782 and Kellof, G. J., et al., Clin. Cancer Res. 11 (2005) 2785- 2808): complete metabolicresponse (CMR) or partial metabolic response (PMR), stable metabolicdisease (SMD) and progressive metabolic disease (PMD) (see Table 2).

TABLE 1 CT-Criteria (acc. to RECIST) CT -measurement: Change in sumslongest diameters RECIST Disappearance; CR conformed at 4 weeks (aftertreatment start) 30% decrease; PR confirmed at 4 weeks Neither PR nor PDSD criteria met 20% increase, no CR, PD PR, SD documented beforeincreased disease

TABLE 2 Proposed FDG-PET criteria (acc. to EORTC, see Young H., et al.,Eur J Canc 35 (1999) 1773-1782) Proposed FDG- PET-measurement PETcriteria Complete resolution of CMR 2-[¹⁸F]-Fluoro-2-deoxy- glucose(FDG) tumor uptake Reduction of a minimum PMR of 15-25% of standardizeduptake value (SUV) after one treatment cycle, and of >25% after morethan one treatment cycle Increase of standardized SMD uptake value (SUV)<25% or decrease of SUV <15% No visible increase the extent of FDG tumoruptake Increase of SUV >25% PMD Visible increase of FDG tumor uptake(>20% of longest dimension) Appearance of new FDG uptake in metastaticlesions

Thus, preferably, “Response (RE)” and “Non-Response (NR)” according tothis invention are established based on data acquired by the combinationof computer tomography (CT) and 2-[¹⁸F]-Fluoro-2-deoxyglucose positronemission tomography (FDG-PET) (Kellof, G. J., et al., Clin. Cancer Res.11 (2005) 2785- 2808, and Young H., et al., Eur. J. Canc. 35 (1999)1773-1782) using both the RECIST and FDG-PET criteria described above.Accordingly Response (RE) and Non-Response (NR) according to thisinvention are determined preferably as follows:

Response (RE): CR or PR is established via CT-RECIST criteria (Table 1)and at the same time CMR or PMR is established via FDG-PET (Table 2).Thus Response (RE) means one of the following four cases for combined CTand PET measurement: CR and CMR, PR and PMR, CR and PMR, and PR and CMR.

Non-Response (NR): SD or PD is established via CT-RECIST criteria(Table 1) and at the same time SMD or PMD is established via FDG-PET(Table 2). Thus the following four cases for combined CT and PETmeasurement signify Non-Response (NR): SD and SMD, SD and PMD, PD andSMD, and PD and PMD.

Usually the response is determined at around 3 to 8 weeks, preferably ataround 6 weeks, after treatment start. This response determination isusually repeated at intervals of 4 to 8 weeks, preferably of 6 to 8weeks. When at the first determination a significant response (RE) wasidentified, then a relapse (that means a Non-Response (RE) after thefirst determination) can be determined at earliest at the secondresponse determination.

In this context, the term “patient who has failed prior therapy with ananti-VEGF antibody” refers to a patient, in whom either at the firstresponse determination Non-Response (NR) is established(“Non-Responder”) or at the first response determination Response (RE)is established, and in the second or a subsequent response determinationNon-Response (NR) is established (“Relapser”).

The term “metastasis” according to the invention refers to thetransmission of cancerous cells from the primary tumor to one or moresites elsewhere in a patient causing secondary tumors. A tumor formed bycells that have spread is called a “metastatic tumor” or a “metastasis”.The metastatic tumor contains cells that are like those in the original(primary) tumor. Means to determine if a cancer has metastasized areknown in the art and include tumor marker tests, bone scan, chest X-ray,computed tomography (CT), computerized axial tomography (CAT), molecularresonance imaging (MRI), positron emission tomography (PET), singlephoton emission computed tomography (SPECT), fluorescence imaging (FI),and bioluminescent imaging (BLI) and tumor marker tests (see e.g. Helms,M. W., et al., Contributions to microbiology 13 (2006) 209-231, andPantel, K., et al., J. Nat. Cancer Inst. 91 (1999) 1113-1124).

As used herein, the term “patient” preferably refers to a human in needof treatment to treat cancer, or a precancerous condition or lesion.However, the term “patient” can also refer to non-human animals,preferably mammals such as dogs, cats, horses, cows, pigs, sheep andnon-human primates, among others, that are in need of treatment.

The term “group” refers to a group of patients as well as a sub-group ofpatients.

The term “package insert” refers to instructions customarily included incommercial packages of therapeutic products, which may includeinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The cancer may be, for example, lung cancer, non small cell lung (NSCL)cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or urethra, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,chronic or acute leukemia, lymphocytic lymphomas, neoplasms of thecentral nervous system (CNS), spinal axis tumors, brain stem glioma,glioblastoma multiforme, astrocytomas, schwannomas, ependymomas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenomas, including refractory versions of any of the above cancers, ora combination of one or more of the above cancers. The precancerouscondition or lesion includes, for example, the group consisting of oralleukoplakia, actinic keratosis (solar keratosis), precancerous polyps ofthe colon or rectum, gastric epithelial dysplasia, adenomatousdysplasia, hereditary nonpolyposis colon cancer syndrome (HNPCC),Barrett's esophagus, bladder dysplasia, and precancerous cervicalconditions. In a preferred embodiment, the cancer to be treated is abreast cancer disease. Also in a preferred embodiment, the cancer ischaracterized by an overexpression of the HER2 receptor protein.

DETAILED DESCRIPTION

The invention provides a combined therapy method of treating a breastcancer disease, comprising administering to the patient atherapeutically effective amount of an anti-HER2 antibody and ananti-VEGF antibody wherein the breast cancer disease is characterized byan overexpression of the HER2 receptor protein. More specifically, theinvention provides a method of treating a breast cancer disease in apatient who has failed prior therapy with an anti-VEGF antibody,comprising administering to the patient a therapeutically effectiveamount of an anti-HER2 antibody and an anti-VEGF antibody whereinpreferably the anti-VEGF antibody is bevacizumab, the patient is human;the anti-HER2 antibody is trastuzumab, and wherein preferably the breastcancer disease is characterized by an overexpression of the HER2receptor protein.

The invention further comprises a method of treating a breast cancerdisease in a patient who has failed prior therapy with an anti-VEGFantibody, comprising administering to the patient a therapeuticallyeffective amount of an anti-HER2 antibody while continuing saidanti-VEGF antibody therapy.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing,either partially or completely, the growth of tumors, tumor metastases,or other cancer-causing or neoplastic cells in a patient. The term“treatment” as used herein, unless otherwise indicated, refers to theact of treating.

The phrase “a method of treating” or its equivalent, when applied to,for example, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in a patient,or to alleviate the symptoms of a cancer. “A method of treating” canceror another proliferative disorder does not necessarily mean that thecancer cells or other disorder will, in fact, be eliminated, that thenumber of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of a patient, is nevertheless deemed anoverall beneficial course of action.

The term “patient” as used herein means a mammal, preferably a human.

The term “therapeutically effective amount” or “effective amount” meansthe amount of the subject compound or combination that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician.

The invention further comprises the use of an anti-HER2 antibody and ananti-VEGF antibody for the manufacture of a medicament for treating abreast cancer disease in a patient who has failed prior therapy with ananti-VEGF antibody, comprising administering to the patient atherapeutically effective amount of an anti-HER2 antibody whilecontinuing said anti-VEGF antibody therapy. The antibodies may beadministered separately or simultaneously.

The term “method for manufacturing a medicament” relates to themanufacturing of a medicament for use in the indication as specifiedherein and in particular for use in the treatment of tumors, tumormetastases, or cancer in general. The term relates to the so-called“Swiss-type” claim format in the indication specified.

In the context of this invention, additional other cytotoxic,chemotherapeutic or anti-cancer agents, or compounds that enhance theeffects of such agents may be used in the anti-VEGF antibody plusanti-HER2 antibody combination. Such agents include, for example:alkylating agents or agents with an alkylating action, such ascyclophosphamide (CTX; e.g. cytoxan®), chlorambucil (CHL; e.g.leukeran®), cisplatin (CisP; e.g. platinol®) busulfan (e.g. myleran®),melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM),mitomycin C, and the like; anti-metabolites, such as methotrexate (MTX),etoposide (VP16; e.g. vepesid®), 6-mercaptopurine (6MP), 6-thiocguanine(6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g.Xeloda®), dacarbazine (DTIC), and the like; antibiotics, such asactinomycin D, doxorubicin (DXR; e.g. adriamycin®), daunorubicin(daunomycin), bleomycin, mithramycin and the like; alkaloids, such asvinca alkaloids such as vincristine (VCR), vinblastine, and the like;and other antitumor agents, such as paclitaxel (e.g. taxol®) andpactitaxel derivatives, the cytostatic agents, glucocorticoids such asdexamethasone (DEX; e.g. decadron®) and corticosteroids such asprednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino aciddepleting enzymes such as asparaginase, leucovorin and other folic acidderivatives, and similar, diverse antitumor agents. The following agentsmay also be used as additional agents: arnifostine (e.g. ethyol®),dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g. doxil®),gemcitabine (e.g. gemzar®), daunorubicin lipo (e.g. daunoxome®),procarbazine, mitomycin, docetaxel (e.g. taxoterel, aldesleukin,carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan),10-hydroxy 7-ethyl-camptothecin (SN38), floxuridine, fludarabine,ifosfamide, idarubicin, mesna, interferon beta, interferon alpha,mitoxantrone, topotecan, leuprolide, megestrol, melphalan,mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin,pipobroman, plicamycin, tamoxifen, teniposide, testolactone,thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil.

In the context of this invention, an anti-hormonal agent may be used inthe anti-VEGF antibody plus anti-HER2 antibody combination. As usedherein, the term “anti-hormonal agent” includes natural or syntheticorganic or peptidic compounds that act to regulate or inhibit hormoneaction on tumors. Antihormonal agents include, for example: steroidreceptor antagonists, anti-estrogens such as tamoxifen, raloxifene,aromatase inhibiting 4(5)-imidazoles, other aromatase inhibitors,42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, andtoremifene (e.g. Fareston®); anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; agonists and/or antagonists of glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH) and LHRH (leuteinizing hormone-releasinghormone); the LHRH agonist goserelin acetate, commercially available asZoladex® (AstraZeneca); the LHRH antagonist D-alaninamideN-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-N6-(3-pyridinylcarbonyl)-L-lysyl-N6-(3-pyridinylcarbonyl)-D-lysyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-proline(e.g. Antide®, Ares-Serono); the LHRH antagonist ganirelix acetate; thesteroidal anti-androgens cyproterone acetate (CPA) and megestrolacetate, commercially available as Megace® (Bristol-Myers Oncology); thenonsteroidal anti-androgen flutamide(2-methyl-N-[4,20-nitro-3-(trifluoromethyl)phenylpropanamide),commercially available as Eulexin® (Schering Corp.); the non-steroidalanti-androgen nilutamide,(5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione);and antagonists for other non-permissive receptors, such as antagonistsfor RAR (retinoic acid receptor), RXR (retinoid X receptor), TR (thyroidreceptor), VDR (vitamin-D receptor), and the like.

The use of the cytotoxic and other anticancer agents described above inchemotherapeutic regimens is generally well characterized in the cancertherapy arts, and their use herein falls under the same considerationsfor monitoring tolerance and effectiveness and for controllingadministration routes and dosages, with some adjustments. For example,the actual dosages of the cytotoxic agents may vary depending upon thepatient's cultured cell response determined by using histoculturemethods. Generally, the dosage will be reduced compared to the amountused in the absence of additional other agents.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

In the context of this invention, additional antiproliferative agentsmay be used in the anti-VEGF antibody plus anti-HER2 antibodycombination, including, for example: Inhibitors of the enzyme farnesylprotein transferase and inhibitors of the receptor tyrosine kinasePDGFR, including the compounds disclosed and claimed in U.S. Pat. Nos.6,080,769; 6,194,438; 6,258,824; 6,586,447; 6,071,935; 6,495,564;6,150,377; 6,596,735 and 6,479,513, and International Publication WO01/40217.

In the context of this invention, an effective amount of ionizingradiation may be carried out and/or a radiopharmaceutical may be used inaddition to the anti-VEGF antibody plus anti-HER2 antibody combination.The source of radiation can be either external or internal to thepatient being treated. When the source is external to the patient, thetherapy is known as external beam radiation therapy (EBRT). When thesource of radiation is internal to the patient, the treatment is calledbrachytherapy (BT). Radioactive atoms for use in the context of thisinvention can be selected from the group including, but not limited to,radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57,copper-67, technetium-99, iodine-123, iodine-131, and indium-111. Wherethe EGFR kinase inhibitor according to this invention is an antibody, itis also possible to label the antibody with such radioactive isotopes.

Radiation therapy is a standard treatment for controlling unresectableor inoperable tumors and/or tumor metastases. Improved results have beenseen when radiation therapy has been combined with chemotherapy.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproductivecells in both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (Gy), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsiderations, but the two most important are the location of the tumorin relation to other critical structures or organs of the body, and theextent to which the tumor has spread. A typical course of treatment fora patient undergoing radiation therapy will be a treatment schedule overa 1 to 6 week period, with a total dose of between 10 and 80 Gyadministered to the patient in a single daily fraction of about 1.8 to2.0 Gy, 5 days a week. In a preferred embodiment of this invention thereis synergy when tumors in human patients are treated with thecombination treatment of the invention and radiation. In other words,the inhibition of tumor growth by means of the agents comprising thecombination of the invention is enhanced when combined with radiation,optionally with additional chemotherapeutic or anticancer agents.Parameters of adjuvant radiation therapies are, for example, containedin International Publication WO 99/60023.

The antibodies are administered to a patient according to known methods,by intravenous administration as a bolus or by continuous infusion overa period of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, or intrathecal routes.Intravenous or subcutaneous administration of the antibodies ispreferred.

The amount of anti-VEGF and anti-HER2 antibody administration and thetiming of administration will depend on the type (species, gender, age,weight, etc.) and condition of the patient being treated and theseverity of the disease or condition being treated.

Dosages for administration of the antibodies according to the inventionare about 1 μg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of antibody by one ormore separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to about 100 mg/kg. In apreferred aspect, the antibodies are administered every two to threeweeks, at a dose ranged from about 1 mg/kg to about 15 mg/kg. Apreferred dose for bevacizumab is 5 mg/kg once every 14 days as an IVinfusion until disease progression is detected. A preferred dose fortrastuzumab is a loading dose of 4 mg/kg administered over a 90-minuteperiod and subsequent weekly infusions of 2 mg/kg administered over a30-minute period.

The present invention further provides a kit (pharmaceutical kit)comprising an anti-VEGF antibody (preferably a pharmaceuticalcomposition thereof), an anti-HE2 antibody (preferably a pharmaceuticalcomposition thereof) and a package insert instructing the user of saidcompositions to administer to a patient, having breast cancer diseasewho has failed prior therapy with an anti-VEGF antibody, the anti-VEGFantibody, preferably within pharmaceutical composition and the anti-HER2antibody, preferably within a pharmaceutical composition. In a preferredembodiment, the kit containers may further include a pharmaceuticallyacceptable carrier. The kit may further include a sterile diluent, whichis preferably stored in a separate additional container. The kit mayfurther include a package insert comprising printed instructionsdirecting the use of the combined treatment as a method for a breastcancer disease. Preferably, the pharmaceutical kit will include a firstcontainer storing a pharmaceutical composition comprising an anti-VEGFantibody and a second container storing a pharmaceutical compositioncomprising an anti-HER2 antibody.

Alternatively, the present invention also provides a pharmaceutical kitcomprising a pharmaceutical composition comprising an anti-VEGFantibody, a pharmaceutical composition comprising an anti-HE2 antibody,and a package insert instructing the user of said compositions toadminister to a patient having breast cancer disease, who has failedprior therapy with an anti-VEGF antibody, said anti-VEGF antibodypharmaceutical composition and an anti-HER2 antibody pharmaceuticalcomposition, wherein the anti-VEGF antibody pharmaceutical compositionand the anti-HER2 antibody pharmaceutical composition are packagedeither in a single container or in two separate containers.

The present invention further provides a pharmaceutical composition, inparticular for use in treating a breast cancer disease that has failedprior therapy with anti-VEGF antibody, comprising an anti-HER2 antibodyand an anti-VEGF antibody. Such composition optionally comprisespharmaceutically acceptable carriers and/or excipients, such as thosecommonly known to one of ordinary skill in the art. In a preferredembodiment the anti-VEGF antibody is bevacizumab and the anti-HER2antibody is trastuzumab. The present invention also provides apharmaceutical kit comprising said pharmaceutical composition comprisingsaid anti-HER2 antibody and said anti-VEGF antibody.

The following Experimental details are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that the specific methods and resultsdiscussed are merely illustrative of the invention and are not to beconsidered in any way limited thereto.

Introduction

The current study examined the antitumor activity of the combination ofbevacizumab and trastuzumab after the failure of bevacizumab treatmentalone in human breast xenograft model. Further aims of the study were toexamine the effects of treatment on metastasis.

Test Agents

Trastuzumab was provided as a 25mg/ml stock solution in Histidine-HCl,alpha-alpha Trehalose (60mM), 0.01% Polysorb, pH 6.0 (Herceptin®).Bevacizumab was provided as a 25 mg/ml stock solution in Na-phosphate,alpha-alpha Trehalose (60 mM), 0.01% Polysorb, pH 6.0 (Avastin®). Bothsolutions were diluted appropriately in PBS for injections.

Cell Lines and Culture Conditions

The human breast cancer cell line KPL-4 has been established from themalignant pleural effusion of a breast cancer patient with aninflammatory skin metastasis and overexpresses ErbB family receptors.(Kurebayashi, J., et al., Br. J. Cancer 79 (1999) 707-17) Tumor cellsare routinely cultured in DMEM medium (PAA Laboratories, Austria)supplemented with 10% fetal bovine serum (PAA) and 2 mM L-glutamine(Gibco) at 37° C. in a water-saturated atmosphere at 5% CO2. Culturepassage is performed with trypsin/EDTA 1× (PAA) splitting twice/week.Cell passage P6 was used for in vivo study.

Animals

SCID beige (C.B.-17) mice; age 10-12 weeks; body weight 18-20 g (CharlesRiver, Sulzfeld, Germany) are maintained under specific-pathogen-freecondition with daily cycles of 12 h light/12 h darkness according tointernational guidelines (GV-Solas; Felasa; TierschG). After arrival,animals are housed in the quarantine part of the animal facility for oneweek to get accustomed to new environment and for observation.Continuous health monitoring is carried out on regular basis. Diet food(Alltromin) and water (acidified pH 2.5-3) are provided ad libitum.

Tumor Growth Inhibition Studies In Vivo

Tumor cells were harvested (trypsin-EDTA) from culture flasks (GreinerTriFlask) and transferred into 50 ml culture medium, washed once andresuspended in PBS. After an additional washing step with PBS andfiltration (cell strainer; Falcon 100 μm) the final cell titer wasadjusted to 0.75×10⁸/ml. Tumor cell suspension was carefully mixed withtransfer pipette to avoid cell aggregation. Anesthesia was performedusing a Stephens inhalation unit for small animals with preincubationchamber (plexiglas), individual mouse nose-mask (silicon) and Isoflurane(Pharmacia-Upjohn, Germany) in a closed circulation system. Two daysbefore injection the fur of the animals was shaved. For intra mammaryfat pad (i.f.m.p.) injection, cells were injected orthotopically at avolume of 20 μl into the right penultimate inguinal mammary fat pad ofeach anesthetized mouse. For the orthotopic implantation, the cellsuspension was injected through the skin under the nipple. Tumor cellinjection corresponds to day 1 of the experiment.

Monitoring

Animals were controlled daily for detection of clinical symptoms ofadverse effects. For monitoring throughout the experiment, the bodyweight of the animals was documented two times weekly and the tumorvolume was measured by caliper twice weekly. Primary tumor volume wascalculated according to NCI protocol (TW=1/2ab2, where a and b are longand short diameters of tumor size in mm, Teicher, B., Anticancer drugdevelopment guide, Humana Press 5 (1997) 92). Calculation values weredocumented as mean and standard deviation.

Treatment of Animals

Tumor-bearing mice were randomized when the tumor volume was roughly 100mm³ (n=10 for each group). Each group was closely matched beforetreatment, which began 20 days after tumor cell injection. Vehicle group(group 1) received 10 ml/kg PBS buffer intraperitoneally (i.p.) onceweekly. Trastuzumab (group 2) was administered i.p. at a loading dose of30 mg/kg, followed by once weekly doses of 15 mg/kg (maintenance dose).The anti-VEGF antibody bevacizumab was given i.p. at a dosage of 5 mg/kgtwice weekly (group 3). At day 40, treatment for group 3 was switched toa combination treatment of bevacizumab (5 mg/kg twice weekly i.p.) withtrastuzumab (15 mg/kg once weekly i.p.).

Evaluation of Metastasis

Spread of tumor cells into the lung was determined in sacrificedanimals. Metastasis was measured according to Schneider, T., et al.,Clin. Exp. Metastasis 19 (2002) 571-582. Briefly, lung tissue washarvested and human Alu sequences were quantified by real-time PCR.Higher human DNA levels, quantified by real-time PCR, correspond tohigher levels of metastasis.

Results

The effect of treatment on primary tumor growth is shown in FIG. 1 andTable 3. Tumors in the vehicle group (group 1) grew rapidly and micewere sacrificed 38 days after injection of tumor cells because ofulceration of tumors and the development of clinical symptoms.Monotherapy with trastuzumab (group 2) exerted no significant effect ontumor volume and mice were therefore sacrificed at day 44. Treatmentwith bevacizumab suppressed tumor growth significantly; however, tumorsstarted to regrow around day 44. Combination treatment of bevacizumaband trastuzumab beginning at day 55 resulted in complete inhibition oftumor growth during the duration of the experiment (day 99) andtreatment was well tolerated.

TABLE 3 Antitumor activity of combined trastuzumab and bevacizumabtreatment on tumor growth after bevacizumab treatment failure (data forFIG. 1). Mean tumor volume in mm³ is reported and the standard deviation(SD). trastuzumab + Day Vehicle SD trastuzumab SD bevacizumab SD 20 11831 120 31 119 35 23 150 30 157 57 126 44 27 209 51 164 77 143 67 30 26976 169 82 138 65 34 348 114 214 114 167 76 37 431 138 293 162 181 78 42462 275 172 63 44 547 315 211 65 48 226 68 51 266 78 55 324 103 58 318100 62 248 81 65 232 75 70 209 69 73 224 56 79 213 68 83 173 57 86 17880 90 150 73 93 141 74 97 134 67 99 130 76

The effect of treatment on lung metastasis is shown in FIG. 2 and Table4. The combination of trastuzumab and bevacizumab after bevacizumabtreatment failure resulted in a sharp decrease in metastasis. Levels ofhuman Alu sequences (correlating to invasion of tumor cells intosecondary tissue) are significantly lower in animals treated withcombination therapy at 99 days over vehicle treated animals that weresacrificed at 28 days and over trastuzumab treated animals sacrificed onday 44. This surprising effect on metastasis is in contrast with theeffect seen with other cytotoxic drugs (Geldof, A. A., et al.,Anticancer Res. 8 (1988) 1335-1339; Murphy, J. Clin. Oncol. 11 (1993)199-201, and De Larco, J. E., et al., Cancer Res. 61 (2001) 2857-2861).

TABLE 4 Effect of treatment on lung metastasis. Alu DNA was quantifiedby real-time PCR and is reported for each animal. trastuzumab + Vehicletrastuzumab bevacizumab human 0.224 1.609 0.010 DNA 0.225 0.084 0.010[ng/ml] 0.148 0.586 0.014 0.011 0.055 0.009 0.037 2.919 0.012 0.0580.078 0.010 0.084 2.741 0.041 0.099 0.017 0.010 0.048 0.340 0.016 0.2790.232 0.027 mean 0.1212* 0.8661** 0.098 median 0.0915 0.2861 0.088Statistical significance of combination treatment *p = 0.001 **p =<0.001

What is claimed is:
 1. A combined therapy method of treating a breastcancer disease, comprising administering to the patient atherapeutically effective amount of an anti-HER2 antibody and ananti-VEGF antibody wherein the breast cancer disease is characterized byan overexpression of the HER2 receptor protein.
 2. A method of treatinga breast cancer disease in a patient who has failed prior therapy withan anti-VEGF antibody, comprising administering to the patient atherapeutically effective amount of an anti-HER2 antibody and ananti-VEGF antibody.
 3. The method of claim 2 wherein the anti-VEGFantibody is bevacizumab.
 4. The method of claim 2 wherein the patient ishuman.
 5. The method of claim 2 wherein the anti-HER2 antibody istrastuzumab.
 6. The method of claim 2 wherein the breast cancer diseaseis characterized by an overexpression of the HER2 receptor protein.
 7. Apharmaceutical kit comprising a pharmaceutical composition comprising ananti-VEGF antibody, a pharmaceutical composition comprising an anti-HE2antibody, and a package insert instructing the user of said compositionsto administer to a patient having breast cancer disease, who has failedprior therapy with an anti-VEGF antibody, said anti-VEGF antibodypharmaceutical composition and said anti-HER2 antibody pharmaceuticalcomposition, wherein the anti-VEGF antibody pharmaceutical compositionand the anti-HER2 antibody pharmaceutical composition are packagedeither in a single container or in two separate containers.
 8. Thepharmaceutical kit of claim 7 wherein the anti-VEGF antibody isbevacizumab.
 9. The pharmaceutical kit of claim 7 wherein the anti-HER2antibody is trastuzumab.
 10. A pharmaceutical composition comprising ananti-HER2 antibody and an anti-VEGF antibody useful in the treatment ofa breast cancer disease in a patient which has failed prior therapy withan anti-VEGF antibody.
 11. The pharmaceutical composition of claim 10,wherein the anti-VEGF antibody is bevacizumab and the anti-HER2 antibodyis trastuzumab.